Daisuke Tokita

High PD-L1/CD86 Ratio on Plasmacytoid Dendritic Cells Correlates With Elevated T-Regulatory Cells in Liver Transplant Tolerance

Background. Both dendritic cells (DC) and T-regulatory cells (Treg) have been implicated in regulation of alloimmune responses and transplant tolerance. Methods. We analyzed B7 coregulatory molecule expression on circulating DC subset precursors, together with CD4+CD25hi Foxp3+ Treg by rare event, flow cytometric analysis in operationally tolerant pediatric liver transplant recipients (TOL), those undergoing prospective immunosuppressive drug weaning (PW) or maintenance immunosuppression (MI), and normal healthy individuals (controls). Results. Use of DC subset-specific monoclonal antibodies confirmed elevated precursor plasmacytoid DC/myeloid DC ratios in TOL and PW compared with MI. In addition, Treg frequencies were higher in TOL than in PW and MI, but not controls. While there was no difference in levels of costimulatory and coinhibitory molecules on precursor myeloid DC between the groups, the programmed death ligand-1 (PD-L1=B7-H1):CD86 (B7-2) ratio on precursor plasmacytoid DC was significantly higher in TOL than MI and correlated with the Treg frequency. There was no relation between prednisone or tacrolimus dose or tacrolimus trough level and either the PD-L1/CD86 ratio on plasmacytoid DC or the Treg frequency. Moreover, clinically relevant concentrations of dexamethasone or tacrolimus did not affect these values in short-term culture. Conclusion. These novel findings suggest a possible functional relationship between the enhanced incidence of precursor plasmacytoid DC, their comparatively high relative expression of the coinhibitory molecule PD-L1, and the elevated frequency of Treg in operational liver transplant tolerance.

Dendritic cells, the liver, and transplantation

Interstitial liver dendritic cells (DCs) exhibit phenotypic diversity and functional plasticity. They play important roles in both innate and adaptive immunity. Their comparatively low inherent T cell stimulatory ability and the outcome of their interactions with CD4+ and CD8+ T cells, as well as with natural killer (NK) T cells and NK cells within the liver, may contribute to regulation of hepatic inflammatory responses and liver allograft outcome. Liver DCs migrate in the steady state and after liver transplantation to secondary lymphoid tissues, where the outcome of their interaction with antigen‐specific T cells determines the balance between tolerance and immunity. Systemic and local environmental factors that are modulated by ischemia‐reperfusion injury, liver regeneration, microbial infection, and malignancy influence hepatic DC migration, maturation, and function. Current research in DC biology is providing new insights into the role of these important antigen‐presenting cells in the complex events that affect liver transplant outcome. (HEPATOLOGY 2007.)

Inhibition of Kupffer cell‐mediated early proinflammatory response with carbon monoxide in transplant‐induced hepatic ischemia/reperfusion injury in rats

Proinflammatory responses play critical roles in hepatic ischemia/reperfusion (I/R) injury associating with liver transplantation (LTx), and carbon monoxide (CO) can effectively down‐regulate them. Using wild‐type (WT) to enhanced green fluorescent protein (EGFP)‐transgenic rat LTx with 18‐hour cold preservation in University of Wisconsin solution, this study analyzed the relative contribution of donor and host cells during early posttransplantation period and elucidated the mechanism of hepatic protection by CO. CO inhibited hepatic I/R injury and reduced peak alanine aminotransferase levels at 24 hours and hepatic necrosis at 48 hours. Abundant EGFP+ host cells were found in untreated WT liver grafts at 1 hour and included nucleated CD45+ leukocytes (myeloid, T, B, and natural killer cells) and EGFP+ platelet‐like depositions in the sinusoids. However, reverse transcription polymerase chain reaction (RT‐PCR) analysis of isolated graft nonparenchymal cells (NPCs) revealed that I/R injury‐induced proinflammatory mediators [for example, tumor necrosis factor alpha (TNF‐α), interleukin‐6 (IL‐6), and inducible nitric oxide synthase (iNOS)] were not up‐regulated in purified CD45+ cells of donor or host origin. Instead, TNF‐α and IL‐6 messenger RNA (mRNA) elevation was exclusively seen in isolated CD68+ cells, whereas iNOS mRNA up‐regulation was seen in hepatocytes. Nearly all CD68+ cells at 1 hour after LTx were EGFP− donor Kupffer cells, and CO efficiently inhibited TNF‐α and IL‐6 up‐regulation in the CD68+ Kupffer cell fraction. When graft Kupffer cells were inactivated with gadolinium chloride, activation of inflammatory mediators in liver grafts was significantly inhibited. Furthermore, in vitro rat primary Kupffer cell culture also showed significant down‐regulation of lipopolysaccharide (LPS)‐induced inflammatory responses by CO. Conclusion: These results indicate that CO ameliorates hepatic I/R injury by down‐regulating graft Kupffer cells in early postreperfusion period. The study also suggests that different cell populations play diverse roles by up‐regulating distinctive sets of mediators in the acute phase of hepatic I/R injury. (HEPATOLOGY 2008;48:1608–1620.)

Endotoxin modulates the capacity of CpG‐activated liver myeloid DC to direct Th1‐type responses

DC are believed to play important roles in the induction and regulation of immune responses in the liver, an organ implicated in peripheral tolerance. Since the liver is located downstream of the gut, it is constantly exposed to bacterial LPS. Our recent observations indicate that prior exposure to endotoxin modulates subsequent liver DC responses to this TLR4 ligand. In this study, we demonstrate that endotoxin modifies the capacity of mouse liver myeloid DC (MDC) activated by CpG (TLR9 ligand) to direct Th1‐type responses. IL‐12 production by liver MDC was significantly lower than that of spleen MDC following CpG or Imiquimod (R837; TLR7 ligand) activation in vitro. In addition, allogeneic T cells stimulated by CpG‐activated liver MDC secreted significantly lower levels of IFN‐γ than T cells stimulated with CpG‐activated spleen MDC. A similar effect on liver DC was observed in response to in vivo CpG administration. This effect may be explained by exposure of the DC to endotoxin, because LPS attenuated IL‐12 production by CpG‐stimulated liver MDC, both in vitro and in vivo. Moreover, attenuation of the response to CpG was not observed in liver MDC from TLR4‐mutant (C3H/HeJ) mice, in which TLR4 signaling is impaired. These data suggest that endotoxin‐induced ‘cross‐tolerance’ to TLR ligands in liver DC may contribute to down‐regulation of hepatic immune responses.

Infusion of stably immature monocyte-derived dendritic cells plus CTLA4Ig modulates alloimmune reactivity in rhesus macaques.

Background. Immature dendritic cells (DC) can promote long-term transplant survival in rodents. We assessed the impact of stably immature, donor-derived DC on alloimmune reactivity in rhesus macaques. Methods. CD14+ monocytes isolated from leukapheresis products of Macacca mulatta were cultured in granulocyte-macrophage colony stimulating factor plus interleukin (IL)-4±vitamin (vit) D3, and IL-10. Major histocompatibility complex class II and cosignaling molecule expression was determined on CD11c+ cells by flow cytometry. T-cell allostimulatory capacity of the DC, including DC exposed to proinflammatory cytokines, was determined in mixed leukocyte reaction. To test their influence in vivo, purified DC were infused intravenously into allogeneic recipients, either alone or followed by CTLA4Ig, 24 hr later. Proliferative responses of recipient CFSE-labeled T cells to donor or third party DC, cytokine production by stimulated T cells, and circulating alloantibody levels were determined by flow cytometry, up to 100 days postinfusion. Results. VitD3/IL-10-conditioned, monocyte-derived DC were resistant to maturation and failed to induce allogeneic T cell proliferation in vitro. After their infusion, an increase in anti-donor and anti–third party T-cell reactivity was observed, that subsequently subsided to fall significantly below pretreatment levels (by day 56) only in animals also given CTLA4Ig. No increase in circulating immunoglobulin (Ig) M or IgG anti-donor alloantibody titers compared with pretreatment values was detected. With DC+CTLA4Ig infusion, alloreactive IL-10-producing T cells were prevalent in the circulation after day 28. Conclusions. Maturation-resistant rhesus DC infusion is well-tolerated. DC+CTLA4Ig infusion modulates allogeneic T-cell responses and results in hyporesponsiveness to donor and third party alloantigens.

Suppressive efficacy and proliferative capacity of human regulatory T cells in allogeneic and xenogeneic responses.

Background. An understanding of the mechanisms that suppress the human anti-pig cellular response is key for xenotransplantation. We have compared the ability of human regulatory T cells (Tregs) to suppress xenogeneic and allogeneic responses in vitro. Methods. Human peripheral blood mononuclear cells (PBMC), CD4+T cells, or CD4+CD25−T cells were stimulated with irradiated human or wild type (WT) or &agr;1,3-galactosyltransferase gene-knockout (GT-KO) pig PBMC in the presence or absence of human CD4+CD25highTregs. In separate experiments, 5- (and 6)-carboxyfluorescein diacetate succinimidyl ester-labeled human CD4+T cells were stimulated with human or pig PBMC. The expansion and precursor frequencies of allo- and xeno-reacitve Tregs were assessed by labeling with FoxP3 mAb and flow cytometric analysis. Results. The responses of human PBMC, CD4+T cells, and CD4+CD25−T cells to pig PBMC were stronger than to human PBMC (P<0.05). Human anti-GT-KO responses were weaker than anti-WT responses (P<0.05). Human CD4+CD25highTregs suppressed proliferation of CD4+CD25−T cells to both human and pig PBMC stimulator cells with the same efficiency. Alloreactive CD4+CD25+FoxP3high responder T cells proliferated more than their xenoreactive counterparts (P<0.05), although xenoreactive CD4+CD25+T cells proliferated more than alloreactive cells (P<0.05). There was no difference in precursor frequency between allo- and xeno-reactive CD4+CD25+FoxP3high cells. Conclusions. Human T-cell responses to pig cells are stronger than to allogeneic cells. The human response to GT-KO PBMC is weaker than to WT PBMC. Although human Tregs can suppress both responses, expansion of CD4+CD25+FoxP3high cells against pig PBMC is weaker than against human PBMC. More human Tregs may be required to suppress the stronger xenogeneic response.

Poor allostimulatory function of liver plasmacytoid DC is associated with pro-apoptotic activity, dependent on regulatory T cells.

BACKGROUND/AIMS The liver is comparatively rich in plasmacytoid (p) dendritic cells (DC), - innate immune effector cells that are also thought to play key roles in the induction and regulation of adaptive immunity. METHODS Liver and spleen pDC were purified from fms-like tyrosine kinase ligand-treated control or lipopolysaccharide-injected C57BL/10 mice. Flow cytometric and molecular biologic assays were used to characterize their function and interaction with naturally occurring regulatory T cells (Treg). RESULTS While IL-10 production was greater for freshly isolated liver compared with splenic pDC, the former produced less bioactive IL-12p70. Moreover, liver pDC expressed a low Delta4/Jagged1 Notch ligand ratio, skewed towards T helper 2 cell differentiation/cytokine production, and promoted allogeneic CD4(+)T cell apoptosis. T cell proliferation in response to liver pDC was, however, enhanced by blocking IL-10 function at the initiation of cultures. In the absence of naturally occurring CD4(+)CD25(+) regulatory T cells, similar levels of T cell proliferation were induced by liver and spleen pDC and the pro-apoptotic activity of liver pDC was reversed. CONCLUSIONS The inferior T cell allostimulatory activity of in vivo-stimulated liver pDC may depend on the presence and function of Treg, a property that may contribute to inherent liver tolerogenicity.

Sphingosine 1-phosphate receptor agonism impairs skin dendritic cell migration and homing to secondary lymphoid tissue: association with prolonged allograft survival.

The novel immunomodulator FTY720 is a prototypic sphingosine-1-phosphate (S1P) receptor agonist that regulates lymphocyte migration and prolongs allograft survival. Skin dendritic cells (DC) play important roles in cutaneous immunity. We investigated the migration and function of skin DC exposed to FTY720 in vivo, or to its metabolite FTY720 phosphate (P) in vitro. C57BL/10 (H2(b)) recipient (but not donor) FTY720 treatment prolonged median skin C3H (H2(k)) allograft survival significantly, from 12 to 34.5 days. Non-transplanted, FTY720-treated mice revealed a marked increase in skin DC, although total DC in skin-draining lymph nodes (DLN) were unchanged compared with controls. Fewer allogeneic donor DC migrated to DLN of FTY720-treated graft recipients. DC that migrated from the skin of FTY720-treated mice showed reduced MHC class II, CD86 and CCR7 expression, suggesting impaired migratory potential to secondary lymphoid tissue, that correlated with DC retention in skin, and reduced T cell stimulatory activity. Fewer DC migrated from normal skin explants exposed to the FTY720 metabolite, FTY720P than to control medium. DC that did migrate expressed lower levels of MHC class II, CD86 and CCR7, and inferior T cell stimulatory ability. These data demonstrate S1P signaling regulates skin DC trafficking and modulates MHC class II, costimulatory, and homing receptor molecule expression that impairs their ability to elicit allogeneic T cell responses.

Allostimulatory activity of bone marrow-derived plasmacytoid dendritic cells is independent of indoleamine dioxygenase but regulated by inducible costimulator ligand expression

We investigated the role of two key immunoregulatory molecules, indoleamine dioxygenase (IDO) and inducible costimulator ligand (ICOSL), in determining the function of bone marrow (BM)-derived plasmacytoid (p)DC, which offer the potential for therapy of allograft rejection. pDC generated from BM of wild-type (WT) or IDO knockout (KO) C57BL/6 mice were used to stimulate T-cell proliferation and interferon-gamma (IFN-gamma) production in response to alloantigen (alloAg) via the direct or indirect pathways. In some experiments, pDC were first activated by exposure to CpG +/- CTLA4Ig for IDO induction via B7 ligation. Although IDO KO pDC induced enhanced T-cell responses compared with WT pDC, the use of the IDO inhibitor 1-methyltryptophan (1-MT) demonstrated that the inferior stimulatory capacity of WT pDC was not caused by the production of functional IDO, even under IDO-inducing conditions. The DNAX-activating protein of 12 kDa (DAP12), which inhibits functional IDO expression, was expressed in BM-pDC. DAP12 silencing increased the T-cell stimulatory capacity of WT pDC, but only in the presence of 1-MT. Compared with WT pDC, activated IDO KO DC expressed much lower levels of ICOSL. Moreover, when ICOSL was blocked on WT pDC, T-cell proliferation resembled that induced by IDO KO pDC, and interleukin (IL)-10 secretion in MLR was markedly decreased. These findings implicate ICOSL-induced IL-10, but not IDO in the regulation of BM-derived pDC function.